Draft Genome Sequence of Staphylococcus aureus 118 (ST772), a Major Disease Clone from India

We report the draft genome sequence of an ST772 Staphylococcus aureus disease isolate carrying staphylococcal cassette chromosome mec (SCCmec) type V from a pyomyositis patient. Our de novo short read assembly is similar to 2.8 Mb and encodes a unique Panton-Valentine leukocidin (PVL) phage with structural genes similar to those of phi 7247PVL and novel lysogenic genes at the N termini.

Clonal complexes and virulence factors of Staphylococcus aureus from several cities in India

Background: Diseases from Staphylococcus aureus are a major problem in Indian hospitals and recent studies point to infiltration of community associated methicillin resistant S. aureus (CA-MRSA) into hospitals. Although CA-MRSA are genetically different from nosocomial MRSA, the distinction between the two groups is blurring as CA-MRSA are showing multidrug resistance and are endemic in many hospitals. Our survey of samples collected from Indian hospitals between 2004 and 2006 had shown mainly hospital associated methicillin resistant Staphylococcus aureus (HA-MRSA) carrying staphylococcal cassette chromosome mec (SCCmec) type III and IIIA. But S. aureus isolates collected from 2007 onwards from community and hospital settings in India have shown SCCmec type IV and V cassettes while several variations of type IV SCCmec cassettes from IVa to IVj have been found in other parts of the world. In the present study, we have collected nasal swabs from rural and urban healthy carriers and pus, blood etc from in patients from hospitals to study the distribution of SCCmec elements and sequence types (STs) in the community and hospital environment. We performed molecular characterization of all the isolates to determine their lineage and microarray of select isolates from each sequence type to analyze their toxins, virulence and immune-evasion factors. Results: Molecular analyses of 68 S. aureus isolates from in and around Bengaluru and three other Indian cities have been carried out. The chosen isolates fall into fifteen STs with all major clonal complexes (CC) present along with some minor ones. The dominant MRSA clones are ST22 and ST772 among healthy carriers and patients. We are reporting three novel clones, two methicillin sensitive S. aureus (MSSA) isolates belonging to ST291 (related to ST398 which is live stock associated), and two MRSA clones, ST1208 (CC8), and ST672 as emerging clones in this study for the first time. Sixty nine percent of isolates carry Panton-Valentine Leucocidin genes (PVL) along with many other toxins. There is more diversity of STs among methicillin sensitive S. aureus than resistant ones. Microarray analysis of isolates belonging to different STs gives an insight into major toxins, virulence factors, adhesion and immune evasion factors present among the isolates in various parts of India. Conclusions: S. aureus isolates reported in this study belong to a highly diverse group of STs and CC and we are reporting several new STs which have not been reported earlier along with factors influencing virulence and host pathogen interactions.

Draft Genome Sequence of Staphylococcus aureus ST672, an Emerging Disease Clone from India

We report the draft genome sequence of methicillin-resistant Staphylococcus aureus (MRSA) strain ST672, an emerging disease clone in India, from a septicemia patient. The genome size is about 2.82 Mb with 2,485 open reading frames (ORFs). The staphylococcal cassette chromosome mec (SCCmec) element (type V) and immune evasion cluster appear to be different from those of strain ST772 on preliminary examination.

Structure of an amidohydrolase, SACOL0085, from methicillin-resistant Staphylococcus aureus COL

Staphylococcus aureus is an opportunistic pathogen that rapidly acquires resistance to frontline antibiotics. The characterization of novel protein targets from this bacterium is thus an important step towards future therapeutic strategies. Here, the crystal structure of an amidohydrolase, SACOL0085, from S. aureus COL is described. SACOL0085 is a member of the M20D family of peptidases. Unlike other M20D peptidases, which are either monomers or dimers, SACOL0085 adopts a butterfly-shaped homotetrameric arrangement with extensive intersubunit interactions. Each subunit of SACOL0085 contains two Mn2+ ions at the active site. A conserved cysteine residue at the active site distinguishes M20D peptidases from other M20 family members. This cysteine, Cys103, serves as bidentate ligand coordinating both Mn2+ ions in SACOL0085.

Genome sequencing unveils a novel sea enterotoxin-carrying PVL phage in staphylococcus aureus ST772 from India

Staphylococcus aureus is a major human pathogen, first recognized as a leading cause of hospital-acquired infections. Community-associated S. aureus (CA-SA) pose a greater threat due to increase in severity of infection and disease among children and healthy adults. CA-SA strains in India are genetically diverse, among which is the sequence type (ST) 772, which has now spread to Australia, Europe and Japan. Towards understanding the genetic characteristics of ST772, we obtained draft genome sequences of five relevant clinical isolates and studied the properties of their PVL-carrying prophages, whose presence is a defining hallmark of CA-SA. We show that this is a novel prophage, which carries the structural genes of the hlb-carrying prophage and includes the sea enterotoxin. This architecture probably emerged early within the ST772 lineage, at least in India. The sea gene, unique to ST772 PVL, despite having promoter sequence characteristics typical of low expression, appears to be highly expressed during early phase of growth in laboratory conditions. We speculate that this might be a consequence of its novel sequence context. The crippled nature of the hlb-converting prophage in ST772. suggests that widespread mobility of the sea enterotoxin might be a selective force behind its `transfer' to the PVL prophage. Wild type ST772 strains induced strong proliferative responses as well as high cytotoxic activity against neutrophils, likely mediated by superantigen SEA and the PVL toxin respectively. Both proliferation and cytotoxicity were markedly reduced in a cured ST772 strain indicating the impact of the phage on virulence. The presence of SEA alongside he genes for the immune system-modulating PVL toxin may contribute to the success and virulence of ST772.

Crystallization and preliminary X-ray diffraction studies of Staphylococcus aureus homoserine dehydrogenase

Staphylococcus aureus is a Gram-positive nosocomial pathogen. The prevalence of multidrug-resistant S. aureus strains in both hospital and community settings makes it imperative to characterize new drug targets to combat S. aureus infections. In this context, enzymes involved in cell-wall maintenance and essential amino-acid biosynthesis are significant drug targets. Homoserine dehydrogenase (HSD) is an oxidoreductase that is involved in the reversible conversion of l-aspartate semialdehyde to l-homoserine in a dinucleotide cofactor-dependent reduction reaction. HSD is thus a crucial intermediate enzyme linked to the biosynthesis of several essential amino acids such as lysine, methionine, isoleucine and threonine.

Novel Rearrangements in the Staphylococcal Cassette Chromosome Mec Type V Elements of Indian ST772 and ST672 Methicillin Resistant Staphylococcus aureus Strains

Staphylococcus aureus is a commensal gram positive bacteria which causes severe and non severe infections in humans and livestock. In India, ST772 is a dominant and ST672 is an emerging clone of Staphylococcus aureus. Both cause serious human diseases, and carry type V SCCmec elements. The objective of this study was to characterize SCCmec type V elements of ST772 and ST672 because the usual PCR methods did not amplify all primers specific to the type. Whole genome sequencing analysis of seven ST772 and one ST672 S. aureus isolates revealed that the SCCmec elements of six of the ST772 isolates were the smallest of the extant type V elements and in addition have several other novel features. Only one ST772 isolate and the ST672 isolate carried bigger SCCmec cassettes which were composites carrying multiple ccrC genes. These cassettes had some similarities to type V SCCmec element from M013 isolate (ST59) from Taiwan in certain aspects. SCCmec elements of all Indian isolates had an inversion of the mec complex, similar to the bovine SCCmec type X. This study reveals that six out of seven ST772 S. aureus isolates have a novel type V (5C2) SCCmec element while one each of ST772 and ST672 isolates have a composite SCCmec type V element (5C2&5) formed by the integration of type V SCCmec into a MSSA carrying a SCC element, in addition to the mec gene complex inversions and extensive recombinations.

Influence of the AgrC-AgrA Complex on the Response Time of Staphylococcus aureus Quorum Sensing

The Staphylococcus aureus agr quorum-sensing system plays a major role in the transition from the persistent to the virulent phenotype. S. aureus agr type I to IV strains are characterized by mutations in the sensor domain of the histidine kinase AgrC and differences in the sequences of the secreted autoinducing peptides (AIP). Here we demonstrate that interactions between the cytosolic domain of AgrC (AgrC(Cyto)) and the response regulator domain of AgrA (AgrA(RR)) dictate the spontaneity of the cellular response to AIP stimuli. The crystal structure of AgrC(Cyto) provided a basis for a mechanistic model of AgrC-AgrA interactions. This model enabled an analysis of the biochemical and biophysical parameters of AgrC-AgrA interactions in the context of the conformational features of the AgrC-AgrA complex. This analysis revealed distinct sequence and conformational features that determine the affinity, specificity, and kinetics of the phosphotransfer reaction. This step, which governs the response time for transcriptional reengineering triggered by an AIP stimulus, is independent of the agr type and similar for agonist and antagonist stimuli. These experimental data could serve as a basis on which to validate simulations of the quorum-sensing response and for strategies that employ the agr quorum-sensing system to combat biofilm formation in S. aureus infections.

Levels of Alpha-Toxin Correlate with Distinct Phenotypic Response Profiles of Blood Mononuclear Cells and with agr Background of Community-Associated Staphylococcus aureus Isolates

Epidemiological studies of Staphylococcus aureus have shown a relation between certain clones and the presence of specific virulence genes, but how this translates into virulence-associated functional responses is not fully elucidated. Here we addressed this issue by analyses of community-acquired S. aureus strains characterized with respect to antibiotic resistance, ST types, agr types, and virulence gene profiles. Supernatants containing exotoxins were prepared from overnight bacterial cultures, and tested in proliferation assays using human peripheral blood mononuclear cells (PBMC). The strains displayed stable phenotypic response profiles, defined by either a proliferative or cytotoxic response. Although, virtually all strains elicited superantigen-mediated proliferative responses, the strains with a cytotoxic profile induced proliferation only in cultures with the most diluted supernatants. This indicated that the superantigen-response was masked by a cytotoxic effect which was also confirmed by flow cytometry analysis. The cytotoxic supernatants contained significantly higher levels of alpha-toxin than did the proliferative supernatants. Addition of alpha-toxin to supernatants characterized as proliferative switched the response into cytotoxic profiles. In contrast, no effect of Panton Valentine Leukocidin, delta-toxin or phenol soluble modulin alpha-3 was noted in the proliferative assay. Furthermore, a significant association between agr type and phenotypic profile was found, where agrII and agrIII strains had predominantly a proliferative profile whereas agrI and IV strains had a predominantly cytotoxic profile. The differential response profiles associated with specific S. aureus strains with varying toxin production could possibly have an impact on disease manifestations, and as such may reflect specific pathotypes.

Structure of an amidohydrolase, SACOL0085, from methicillin-resistant Staphylococcus aureus COL

Staphylococcus aureus is an opportunistic pathogen that rapidly acquires resistance to frontline antibiotics. The characterization of novel protein targets from this bacterium is thus an important step towards future therapeutic strategies. Here, the crystal structure of an amidohydrolase, SACOL0085, from S. aureus COL is described. SACOL0085 is a member of the M20D family of peptidases. Unlike other M20D peptidases, which are either monomers or dimers, SACOL0085 adopts a butterfly-shaped homotetrameric arrangement with extensive intersubunit interactions. Each subunit of SACOL0085 contains two Mn2+ ions at the active site. A conserved cysteine residue at the active site distinguishes M20D peptidases from other M20 family members. This cysteine, Cys103, serves as bidentate ligand coordinating both Mn2+ ions in SACOL0085.

Utilización de la resina de neem (Azadirachta indica) como desparasitante externo en el tratamiento del tórsalo (Dermatobia hominis) en bovinos del municipio de Muy Muy, departamento de Matagalpa

El presente estudio se realizó con el objetivo de evaluar la utilización de la Resina de Neem (Azadirachta indica)como desparasitante externo en el tratamiento del tórsalo (Dermatobia hominis)en bovinos del Municipio de Muy Muy, Departamento de Matagalpa. El Municipio de Muy Muy esta ubicado en las Coordenadas 12º45'48" de latitud Norte con 85° 37' 36" de Longitud Oeste, con una altura sobre el nivel del mar de 337.6 metros, una precipitación promedio entre 1400-1800 mm, con una temperatura promedio 24°C., La topografía del terreno en que se ubica el Municipio de MUY MUY, presenta las siguientes características: 32.1% Terrenos Plano, 41.0% Terrenos Ondulados y 26.9% Terrenos Quebrados. Y los tipos de suelos son arcillosos y rocosos. En el trabajo experimental se utilizó un diseño completamen te al azar (D.C.A) el que estuvo compuesto por un lote de 18 animales divididos en 3 grupos, cada grupo formado por 6 animales seleccionadas al azar y sometidas a tratamientos distintos Tratamiento I: Ivermectina al 1%, Tratamiento II: Resina del Neem al 5%. Tratamiento III: Resina de Neem al 10%. Para evaluar la efectividad de los distintos tratamientos se hizo conteos periódicos de tórsalos cada siete días, cuantificando la cantidad de nódulos por animal y región anatómica a los 7, 14, 21, 28, 35, 42, 49, 56 días. La efectividad de todos los tratamientos se observó a partir de los 21 días después de la aplicación. Los tratamientos II y I tuvieron las mejores respuestas en el control, del tórsalo, con un porcentaje de efectividad del 91.6%, y 72.7% respectivamente y con un 62.5% para el tratamiento III. El área anatómica mas afectada fue la del miembro anterior derecho seguido del lateral derecho. Según el análisis del costo de la dosis, se puede decir que la resina de Neem es un desparasita nte económico para los productores.

Determinar la efectividad del nosodes homeopático en el control del Dermatobia hominis (tórsalo) en la unidad de producción Balcar en el departamento de Esteli

El Dermatobia hominis (torsalo) es una de las patologías que mas afecta al ganado bovino, causa grandes perdidas económicas al productor así como a la economía del país. Con objetivo de brindarle al productor una alternativa para el tratamiento y control del torsalo se desarrollo en el departamento de Estelí el estudio” DETERMINAR LA EFECTIVIDAD DEL NOSODES HOMEOPÁTICO EN EL CONTROL DEL DERMATOBIA HOMINIS (TORSALO)EN LA UNIDAD DE PRODUCCIÓN BALCAR EN EL DEPARTAMENTO DE ESTELÍ. ”Para lo cual se seleccionaron cuarenta animales de forma aleatoria, estos eran terneros de tres a seis meses a los cuales se les aplico medicamento orgánico (nosodes homeopático)durante tres meses (junio, julio y agosto del 2005)donde se aplicaron cuatro tipos de tratamientos . Los resultados obtenidos revelan que el nosodes por digestión sin aditivos(A)y el nosodes por congelación mas aditivos (D) presentan mejores resultados positivos en cuanto a la reducción del ectoparásito.

Estudio epidemiológico de la prevalencia de tórsalo (Dermatobia hominis) en bovinos en San Pedro de Lóvago, Chontales, Nicaragua

El presente estudio se realizó con el objetivo de determinar la prevalencia del tórsalo (Dermatobia hominis) en fincas de las diferentes comarcas ubicadas en el Municipio de San Pedro de Lóvago, departamento de Chontales. El municipio se localiza entre las coordenadas 12º 07 ́ latitud norte y 85º07 ́ latitud oeste. La altitud promedio es de 340msnm. El clima es semi húmedo conocido como de sabana tropical. La temperatura promedio anual oscila entre los 25 y 26ºC; su precipitación pluvial varía entre los 1 200 y 1 400mm, caracterizándose por una buena distribución de las lluvias durante todo el año. En el trabajo de muestreo se utilizó un estudio observacional de tipo transversal, utilizándose un muestreo sin reemplazo. Para determinar el tamaño de la muestra se utilizó la formula de Martin et al. (1987) y Trusfield, (1995) donde plantean que N = 1,962* p *q / L 2,donde p es la prevalencia, q = 1 – p y L especifica el límite deseado de error de la prevalencia. Se espera que la prevalencia (p) de 50% sea usada en combinación con él límite deseado de error de 14%, si la prevalencia en la población entera es desconocida. Y cuando la población de bovinos está comprendida entre los rangos de 2 807 a 7 480 individuos de la población. El tamaño requerido de la muestra de este trabajo fue de 52 fincas en producción de toda la zona en estudio. Los resultados obtenidos en el municipio de San Pedro de Lóvago en relación a la Prevalencia de tórsalo en bovinos, de un total de 52 fincas estudiadas, de los cuales se examinaron un total 1 317 animales, de los cuales 1 068 resultaron positivos representando el 81% de prevalencia y 249 animales negativos para un 19%. Al realizar el análisis estadístico se encontró diferencia significativa (p <0.05) entre las regiones anatómicas, siendo las regiones Laterales Toráxico-abdominal derecha, la más afectada. Al relacionar la intensidad de invasión del tórsalo de acuerdo al color de la piel, se observó que existe mayor presentación del mismo en los animales de color Negro, seguido del color Osca, Blanco y color Pardo.

Prueba de dos insecticidas sistémicos en dos formas de aplicación, en el control del tórsalo (Dermatobia hominis) Lin. Jr

Con el objeto de determinar cuál es el insecticida más efectivo en el control del tórsalo y la mejor forma de aplicación, se llevo a cabo un experimento en el Departamento de Boaco en el lapso comprendido de Julio a Diciembre de 1967. En cada finca se utilizaron cuarenta animales. Los insecticidas aplicados fueron: Ruelene 25 E en dosis de diez y siete miligramos por kilogramo de peso vivo y Neguvón en dosis de cincuenta miligramos por kilogramo de peso vivo. Los insecticidas se aplicaron en la forma siguiente: Ruelene 25 E vertido Ruelene 25 E solución Neguvón vertido Neguvón solución Se efectuaron recuentos de parásitos al principio del experimento y a los tres días y a los ocho días después de la aplicación de los insecticidas. El diseño experimental usado fue de bloques al azar con repeticiones y análisis dentro de las fincas. Los dos insecticidas y sus formas de aplicación estadísticamente tuvieron la misma efectividad, con porcentajes de control similares. el ruelene 25 E fue el insecticida de menor costo, en las dos formas de aplicación. Durante el experimento no se observaron efectos tóxicos en ningún animal. Se observó que el poder residual del Ruelene 25 E fue mayor que el del Neguvón.